Throughout this application, various publications are referenced within parentheses. Disclosures of these publications in their entireties are hereby incorporated by reference into this application to more fully describe the state of the art to which this invention pertains. Full bibliographic citations for these references may be found immediately preceding the claims.
Kaposi""s sarcoma-associated herpesvirus (KSHV) or human herpesvirus 8 (HHV8) is the most recently described DNA tumor virus. It is the infectious trigger for Kaposi""s sarcoma, body cavity-based primary effusion lymphomas (PEL), and some subtypes of multicentric Castleman""s disease (CD) for review see (37), KSHV-related CD is a polyclonal B cell hyperplasia that is presumably driven by KSHV vIL-6 secretion as well as other viral proteins. In contrast, PEL are B cell lymphomas that generally have a monoclonal origin as determined by immunoglobulin gene rearrangement and viral terminal repeat analyses (7, 20, 36). Terminal repeat analyses by Judde and colleagues (20) have also demonstrated that KS tumors can have an oligo- or monoclonal pattern, and may evolve from a polyclonal hyperplasia into a monoclonal tumor. Thus, KSHV may contribute to cell proliferation through secretion of viral cytokines and induction of cellular cytokines as in the case of CD, as well as through expression of transforming viral oncogenes, particularly in the case of PEL.
The KSHV genome has significant sequence homology to all classes of herpesviruses, but is unique among the human herpesviruses in encoding an extensive number of regulatory genes which have been pirated from the host genome during its evolution (30, 36). While a number of these genes have homology to known cellular oncogenes or transform rodent cell lines in vitro (2, 14, 26), only a small number of KSHV genes are routinely found to be expressed in tumor tissues. vBCL-2, vIRF1, vGPCR, and K01 are examples of KSHV proteins which might contribute to cell transformation in vitro but are not appreciably expressed in most KSHV-infected KS or PEL tumors (21, 24, 32, 38).
KSHV infected PEL cell lines constitutively express three viral genes, vFLIP (ORFK13), vCYC (ORF72), and LANA1 (ORF73), which are not inducible by tetradecanoyl phorbol acetate (TPA) or inhibited by phosphonoformic acid (PFA) and thus are unambiguously designated as latent or class I genes. These three proteins are transcribed on the major polycistronic latent transcripts, LT1 and LT2 (10, 39, 42). In vitro studies demonstrate that the viral cyclin associates with cyclin dependent kinase (CDK) 4 and 6, and phosphorylates pRB (8, 16, 28). LANA1 is believed to bind to the origin of replication to tether the viral genome to host chromatin during mitosis, effecting equal segregation of viral genome during division (3). LANA1 also binds to p53 and inhibits p53-mediated transcriptional activity and apoptosis (13). vCYC over-expression induces apoptosis (31) and it is at least theoretically possible that this may be inhibited in situ by the anti-apoptotic activities of other latency expressed proteins, such as vFLIP and LANA1.
Viral protein expression is highly restricted in KS and PEL tumors. Presently, only LANA1 protein has been shown by immunohistochemistry to be expressed in situ in all cells infected by KSHV (11, 22, 32). Viral cyclin and ORFK12 transcripts have been identified by in situ hybridization in all KSHV infected cells (9, 34), however, protein localization has yet to be performed. No other viral proteins examined thus far, including vIL-6 (K2), minor capsid protein (ORF26), K8, K8.1, vIRF1 (K9), K10, k11, PF-8 (ORF59), and ORF65 have a similar in situ constitutive pattern of expression (21, 32).
KSHV gene expression studies remain controversial. Since PEL cell lines can be manipulated into lytic replication by TPA and butyrate, studies on cultured cell lines have been used to classify KSHV genes into mutually-exclusive latent and lytic classes based on transcription kinetics (40). Frequently, KSHV expression patterns from cultured cell studies are assumed to be similar in tumor tissues in situ without direct evidence. However, a number of KSHV genes are expressed at low levels in resting PEL cell lines but are induced to high expression levels during TPA treatment and thus have properties of both latent and lytic genes (analogous to the EBV LMP1 expression pattern). This pattern of gene expression has been referred to as class II expression (37). Recent studies demonstrate that extension of results from expression studies in tissue culture cannot be uniformly applied to human tumor tissues in part because KSHV may have tissue-specific gene expression patterns. vIL-6, for example, behaves as a class II protein in tissue culture cell lines and is expressed in hematopoietic-derived cells but generally not in KS lesions (29). Thus, determining precisely which viral genes are likely to play a role in KSHV-related pathogenesis requires direct tissue examination of each tumor type. Discovery of additional genes that are constitutively expressed in KSHV-induced disorders is particularly important since these genes are likely to play a role in cell growth dysregulation.
For these reasons, discovery of a KSHV gene having a tissue-specific expression profile is important, particularly if the encoded protein is functionally capable of contributing to cell proliferation. In this paper we describe a new KSHV gene (K10.5) expressed in KSHV-infected hematopoietic tissues. This gene is located in a region containing a cluster of viral sequences with limited homology to the interferon regulatory factor (IRF) family of proteins (36). vIRF1 is encoded by ORF K9 and inhibits interferon-induced transcription and fully transforms NIH3T3 cells (12, 14, 27, 44). vIRF1 binds to histone acetyltransferase transcriptional coadaptors (5, 19) and induces cell transformation by activating the cMYC oncogene through an interferon-stimulated response element (ISRE) called the PRF element (19). Based on these findings and the fact that other tumor viruses target the same tumor suppressor pathways as KSHV, Jayachandra et al. found that both Epstein-Barr virus (EBV or HHV4) EBNA2 and adenovirus E1A proteins also activate cMYC but use differing sets of coadaptors from those used by vIRF1 (19). vIRF1 additionally inhibits p53- and Fas-induced apoptosis ((5) and unpublished obs, S. Jayachandra, P. S. Moore, Y. Chang). vIRF1, however, is not generally expressed in PEL or KS and is therefore unlikely to contribute to these diseases although it may be important in the pathogenesis of CD (21, 32). Another IRF-like KSHV open reading frame encoding vIRF2 and having NF-kB-inhibitory activity has been described (6). We show here that LANA2 is a B-cell specific factor that antagonizes p53 tumor suppressor functions and is expressed during latency.
KSHV/HHV8 is associated with three proliferative diseases ranging from viral cytokine-induced hyperplasia to monoclonal neoplasia: multicentric Castleman""s disease (CD), Kaposi""s sarcoma (KS), and primary effusion lymphoma (PEL). Here we report a new latency-associated 1704 bp KSHV spliced gene belonging to a cluster of KSHV sequences having homology to the interferon regulatory factor (IRF) family of transcription factors. ORFK10.5 encodes a protein, latency-associated nuclear antigen 2 (LANA2), which is expressed in KSHV-infected hematopoietic tissues including PEL and CD, but not KS lesions. LANA2 is abundantly expressed in the nuclei of cultured KSHV infected B-cells. Transcription of K10.5 in PEL cell cultures is not inhibited by DNA polymerase inhibitors nor significantly induced by phorbol ester treatment. Unlike LANA1, LANA2 does not elicit a serologic response from patients with KS, PEL or CD as measured by Western-blot hybridization. Both KSHV vIRF1 (ORFK9) and LANA2 (ORFK10.5) appear to have arisen through gene duplication of a captured cellular IRF gene. LANA2 is a potent inhibitor of p53-induced transcription in reporter assays. LANA2 antagonizes apoptosis due to p53 overexpression in p53-null SAOS-2 cells and apoptosis due to doxorubicin treatment of wild-type p53 U20S cells. While LANA2 specifically interacts with aminoacids 290-393 of p53 in glutathione-S-transferase pull-down assays, we were unable to demonstrate LANA2-p53 interaction in vivo by immunoprecipitation. These findings show that KSHV has tissue-specific latent gene expression programs and identify a new latent protein which may contribute to KSHV tumorigenesis in hematopoietic tissues via p53 inhibition.
This invention provides an isolated nucleic acid which encodes a Kaposi""s sarcoma-associated herpesvirus latency-associated nuclear antigen 2 polypeptide (LANA2) or a fragment thereof.
This invention provides a replicable vector which comprises the isolated nucleic acid which encodes a Kaposi""s sarcoma-associated herpesvirus latency-associated nuclear antigen 2 polypeptide.
This invention provides a host vector system which comprises the above vector and a suitable host cell. In one embodiment of the above host vector system, the host cell includes but is not limited to a eukaryotic cell, a hematopoietic cell, a B cell, a bacterial cell and E. Coli. 
This invention provides a method of producing a polypeptide which comprises growing the above host vector system under suitable conditions permitting production of the polypeptide and recovering the polypeptide so produced.
This invention further provides an isolated nucleic acid comprising consecutive nucleotides having the sequence of a promoter of Kaposi""s sarcoma-associated herpesvirus latency-associated nuclear antigen 2 transcription.
This invention provides a replicable vector which comprises the isolated nucleic acid comprising consecutive nucleotides having the sequence of a promoter of latency-associated nuclear antigen 2 transcription operably linked to a second nucleic acid.
This invention provides a host vector system which comprises a replicable vector which comprises the nucleic acid comprising consecutive nucleotides having the sequence of a promoter of latency-associated nuclear antigen 2 transcription operably linked to a second nucleic acid and a suitable host cell.
This invention provides a method of producing a polypeptide which comprises growing the above host vector system under suitable conditions permitting production of the polypeptide and recovering the polypeptide so produced.
This invention provides an isolated nucleic acid capable of specifically hybridizing to the isolated nucleic acid which encodes a Kaposi""s sarcoma-associated herpesvirus latency-associated nuclear antigen 2 polypeptide or a fragment thereof.
This invention provides a Kaposi""s sarcoma-associated herpesvirus latency-associated nuclear antigen 2 polypeptide or a fragment thereof.
This invention also provides an antibody capable of specifically binding to the above polypeptide.
This invention provides a composition comprising the above antibody and an agent conjugated to the antibody.
This invention provides a method of determining whether a subject is afflicted with a disease associated with Kaposi""s sarcoma-associated herpesvirus (KSHV) infection of a B cell which comprises: (a) obtaining a suitable sample from the subject; (b) contacting the suitable sample with a detectable antibody capable of binding to Kaposi""s sarcoma-associated herpesvirus latency-associated nuclear antigen 2 polypeptide or a fragment thereof so as to form a complex between the antibody and any Kaposi""s sarcoma-associated herpesvirus latency-associated nuclear antigen 2 polypeptide or a fragment thereof present in the sample;(c) removing any unbound antibody; and (d) detecting any antibody which is bound to any Kaposi""s sarcoma-associated herpesvirus latency-associated nuclear antigen 2 polypeptide or a fragment thereof in the sample, wherein the presence of antibody indicates that the subject is afflicted with the disease associated with Kaposi""s sarcoma-associated herpesvirus infection of a B cell.
This invention provides a method of determining whether a subject is afflicted with a disease associated with Kaposi""s sarcoma-associated herpesvirus infection of a B cell which comprises:(a) obtaining a suitable sample from the subject; (b) immobilizing a capturing antibody wherein the capturing antibody is capable of binding to Kaposi""s sarcoma-associated herpesvirus latency-associated nuclear antigen 2 to a support; (c) removing any unbound capturing antibody; (d) contacting the capturing antibody with the suitable sample so as to form a complex between the antibody and any Kaposi""s sarcoma-associated herpesvirus latency-associated nuclear antigen 2 present in the sample; (e) removing any unbound sample; (f) contacting the complex obtained in step (d) with a detectable antibody of Kaposi""s sarcoma-associated herpesvirus latency-associated nuclear antigen 2 polypeptide or a fragment thereof so as to form a complex between the detectable antibody and the complex; (g) removing any unbound detectable antibody; and (h) detecting any detectable antibody which is bound to the complex wherein the presence of detectable antibody indicates that the subject is afflicted with the disease associated with Kaposi""s sarcoma-associated herpesvirus infection of a B cell.
This invention provides a method of determining whether a subject is infected with Kaposi""s sarcoma-associated herpesvirus which comprises:(a) obtaining a suitable sample from the subject; (b) contacting the suitable sample with the detectable antibody of Kaposi""s sarcoma-associated herpesvirus latency-associated nuclear antigen 2 polypeptide or a fragment thereof so as to form a complex between the antibody and any polypeptide or fragment thereof present in the sample;(c) removing any unbound antibody; and (d) detecting any antibody which is bound to any Kaposi""s sarcoma-associated herpesvirus latency-associated nuclear antigen 2 polypeptide or fragment thereof in the sample, wherein the presence of antibody indicates that the subject is infected with Kaposi""s sarcoma-associated herpesvirus.
This invention provides a method of determining whether a subject is infected with Kaposi""s sarcoma-associated herpesvirus which comprises:(a) obtaining a suitable sample from the subject; (b) immobilizing a capturing antibody wherein the capturing antibody is capable of binding to polypeptide or fragment thereof to a support; (c) removing any unbound capturing antibody;(d) contacting the capturing antibody with the suitable sample so as to form a complex between the antibody and Kaposi""s sarcoma-associated herpesvirus latency-associated nuclear antigen 2 polypeptide or fragment thereof present in the sample; (e) removing any unbound sample; (f) contacting the complex obtained in step (d) with the detectable antibody which is bound to Kaposi""s sarcoma-associated herpesvirus latency-associated nuclear antigen 2 polypeptide or fragment thereof so as to form a complex between the detectable antibody and the complex; (g) removing any unbound detectable antibody; and (h) detecting any detectable antibody which is bound to the complex wherein the presence of detectable antibody indicates that the subject is infected with Kaposi""s sarcoma-associated herpesvirus.
This invention provides a kit for diagnosing Kaposi""s sarcoma-associated herpesvirus infection comprising the labeled antibody capable of specifically binding to the Kaposi""s sarcoma-associated herpesvirus latency-associated nuclear antigen 2 polypeptide or fragment thereof.
This invention provides a method of inhibiting p53 mediated apoptosis of a cell which comprises introducing into the cell an effective amount of the replicable vector which comprises the isolated nucleic acid which encodes Kaposi""s sarcoma-associated herpesvirus latency-associated nuclear antigen 2 polypeptide or fragment thereof, so as to thereby inhibit p53 mediated apostosis of the cell.
This invention provides a method of immortalizing a cell which comprises introducing into the cell an amount of the replicable vector which comprises the isolated nucleic acid which encodes Kaposi""s sarcoma-associated herpesvirus latency-associated nuclear antigen 2 polypeptide or fragment thereof effective to inhibit p53 mediated apoptosis of the cell, so as to thereby immortalize the cell.
This invention provides a method of producing an antibody which comprises introducing into a cell an amount of the replicable vector which comprises the isolated nucleic acid which encodes Kaposi""s sarcoma-associated herpesvirus latency-associated nuclear antigen 2 polypeptide effective to inhibit p53 mediated apoptosis of the cell producing the antibody and thereby immortalizing the cell, so as to thereby produce the antibody.
This invention provides a method of determining whether a subject is infected with Kaposi""s sarcoma-associated herpesvirus which comprises: (a) obtaining a suitable sample from the subject; (b) contacting the suitable sample with a detectable nucleic acid capable of hybriding to a nucleic acid which encodes Kaposi""s sarcoma-associated herpesvirus latency-associated nuclear antigen 2 polypeptide or fragment thereof under hybridizing conditions so as to form a complex between the detectable nucleic acid and any nucleic acid which encodes a Kaposi""s sarcoma-associated herpesvirus latency-associated nuclear antigen 2 polypeptide or fragment thereof which is present in the sample; (c) removing any unbound detectable nucleic acid; and (d) detecting any detectable nucleic acid which is bound to the complex, wherein the presence of detectable nucleic acid indicates that the subject is infected with Kaposi""s sarcoma-associated herpesvirus.
This invention provides a kit for diagnosing Kaposi""s sarcoma-associated herpesvirus infection comprising a labeled nucleic acid which encodes a Kaposi""s sarcoma-associated herpesvirus latency-associated nuclear antigen 2 polypeptide or fragment thereof.
This invention provides a transgenic non-human animal which has stably integrated into the genome of its germ cells or somatic cells an exogenous nucleic acid construct wherein the nucleic acid construct comprises a B-cell specific promoter of Kaposi""s sarcoma-associated herpesvirus latency-associated nuclear antigen 2 operably linked to a second nucleic acid which encodes a gene of interest and is introduced into the transgenic non-human animal, or an ancestor, at an embryonic stage. In one embodiment of the above transgenic animal, the animal is a mammal.
This invention provides a method for evaluating in a non-human transgenic animal the potential therapeutic effect of an agent for treating Kaposi""s sarcoma-associated herpesvirus infection in a human, which comprises: (a) providing an agent to a transgenic non-human animal whose cells comprise the nucleic acid which encodes a Kaposi""s sarcoma-associated herpesvirus latency-associated nuclear antigen 2 polypeptide; and (b) determining the therapeutic effect of the agent on the transgenic non-human animal by monitoring Kaposi""s sarcoma-associated herpesvirus latency-associated nuclear antigen 2 expression, wherein a decrease in Kaposi""s sarcoma-associated herpesvirus latency-associated nuclear antigen 2 indicates that the agent would have a potential therapeutic effect on Kaposi""s sarcoma-associated herpesvirus infection in a human. In one embodiment of the above method, the animal is a mammal.
This invention provides a method of treating Kaposi""s sarcoma-associated herpesvirus infection in a subject, which comprises introducing into the subject""s cells an effective amount of the nucleic acid capable of specifically hybridizing to the isolated nucleic acid which encodes Kaposi""s sarcoma-associated latency-associated nuclear antigen 2 polypeptide or fragment thereof to hybridize to any of the above nucleic acid which is present in the subject""s cells, so as to thereby treat Kaposi""s sarcoma-associated herpesvirus infection.
This invention provides a method of treating Kaposi""s sarcoma-associated herpesvirus infection in a subject, which comprises introducing into the subject""s cells an effective amount of a nucleic acid capable of specifically hybridizing to an isolated nucleic acid comprising nucleotides having the sequence of a promoter of latency-associated nuclear antigen 2 transcription to hybridize to any of this nucleic acid which is present in the subject""s cells, so as to thereby treat the subject.
This invention provides a composition comprising the antibody capable of specifically binding to the polypeptide encoded by the isolated nucleic acid which encodes Kaposi""s sarcoma-associated latency-associated nuclear antigen 2 polypeptide and a carrier.
This invention provides a method of treating a subject infected with Kaposi""s sarcoma-associated herpesvirus, which comprises administering to the subject an amount of the above composition under conditions such that the antibody binds to any LANA2 present in the subject, so as to thereby treat the subject.
This invention provides a composition comprising the polypeptide encoded by the isolated nucleic acid which encodes Kaposi""s sarcoma-associated latency-associated nuclear antigen 2 polypeptide or fragment thereof and a carrier.